Non-Typhoidal Salmonella (NTS) are substantial foodborne pathogens that lead to bacteremia, gastroenteritis, and focal infection. Poultry is one of the usual provenances for the development of multidrug-resistance NTS. This problem has increased in developing countries with the indiscriminate use of antibiotics in the poultry production system. The current study aimed to determine the prevalence and tendency of antimicrobial resistance of zoonotic Salmonella spp. A total of 601 samples, including cloacal samples (150) eggshell (150), egg content (15 pooled samples), layer hen carcasses (150), hand swab (68) and stool samples (68) from poultry workers, were collected from five layer chicken farms. Isolation of NTS was performed by using different cultural and biochemical methods. Moreover, Salmonella isolates were evaluated for antimicrobial susceptibility using the disc diffusion method. The cloacal samples and stool samples showed the prevalence of Salmonella spp. at approximately similar rates of 4.7% and 4.4%, respectively. Chicken isolates were identified as S. Enteritidis, S. Typhimurium, and S. Gallinarum while the human isolates were only S. Typhimurium and S. Enteritidis. The prevalence of the NTS on the surface of the eggshells (7.3%) was higher than that in the other samples. Among 12 antimicrobials tested, 86.4% resistance was found to streptomycin and oxytetracycline followed by neomycin and erythromycin (77.3%), norfloxacin and ampicillin (68.2%) across the study sites. Kanamycin and gentamicin remained sensitive by 95.5% and 90.9%, respectively. The present study indicated that layer chickens and its products are important sources for human infection with multiple-drug resistant NTS strains.